Abstract 141: Targeting protein secretion as a novel therapeutic strategy in AL amyloidosis
Authors: Maria Moscvin, Peter Gregor Czarnecki, Tianzeng Chen, Annamaria Gullá, Kenneth Carl Anderson, Giada Bianchi
Journal: Cancer Research (2022)

Abstract AL amyloidosis (AL) is an incurable plasma cell disorder. The solely pathogenic mechanism in AL is deposition of immunoglobulin free light chains (FLC) organized in fibrils in target organs. Surprisingly, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, which are the specific target of botulinum neurotoxin (BoNT), are involved in the docking and fusion of secretory vesicles. We hypothesized that certain BoNT serotypes may block FLC secretion and trigger cytotoxicity triggering a terminal UPR. Gene expression profiling in a cohort of 170 newly diagnosed multiple myeloma patients (IFM170) was used to interrogate SNAREs expression in malignant plasma cells. We developed tetracycline inducible, bicistronic vectors expressing distinct BoNT serotypes (BoNT/A-F), T2A and GFP. Lentivirally transduced cells would express BoNT in a 1:1 stochiometric ratio with GFP, upon doxycycline (dox) administration. We transduced AL cell lines stably expressing rRTA with Tet-On lentivirus expressing 7 distinct BoNTs and performed two sets of experiments. First, we performed time-course viability assays on polyclonally transduced cells and compared relative proportion of GFP+ cells over time. Then, we single-cell sorted transduced cells, triggered BoNT expression and assessed GFP kinetic and apoptosis at 24, 48 and 72 hours post dox. SNAREs cleavage following induction of BoNT expression was evaluated via WB in GFP+ clones. To assess if BoNT cytotoxicity correlated with cessation of FLC secretion, we performed a secretion assay in monoclones expressing distinct BoNTs. IFM170 GEP and AL/MM cell lines analysis showed VAMP2, VAMP3 and SNAP23 as the top expressed SNAREs. By using polyclonally transduced cells, we show that GFP+ (transduced) cells are rapidly depleted over time after dox, across all serotypes, except BoNT/B, consistent with cytotoxic effect. We noted an association between SNAP23 and VAMP3 cleavage and BoNT toxicity, suggesting that dual targeting of SNAP23/VAMP3 may be necessary to mediate BoNT cytotoxicity. We next show that only BoNTs causing early cytotoxicity (A, D, E and F) significantly inhibited FLC secretion and induced UPR activation, presumably through FLC retention. Indeed, cytotoxic BoNTs, activated PERK pathway with eIF2a phosphorylation (p-eIF2a); ATF4, CHOP and GADD34 upregulation. Importantly preliminary data in CHOP KO AL cells show rescue of BoNT_induced death, confirming that a terminal UPR is the mechanism of cytotoxicity of BoNT in AL. We show that in all seven BoNT serotypes examined, except for BoNT/B, simultaneous cleavage of SNAP23 and VAMP3 predicts BoNT cytotoxicity and correlates with cessation of FLC secretion and terminal UPR. Overall, our preliminary data provide proof of concept that targeting FLC secretion is feasible and of therapeutic efficacy in preclinical AL models, suggesting potential clinical translatability of this innovative approach. Citation Format: Maria Moscvin, Peter Gregor Czarnecki, Tianzeng Chen, Annamaria Gullá, Kenneth Carl Anderson, Giada Bianchi. Targeting protein secretion as a novel therapeutic strategy in AL amyloidosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 141.